The hypothesis to be addressed in this proposal is that activation of the dopamine system is necessary for the psychomotor stimulant properties of nicotine but is not sufficient to support nicotine dependence and withdrawal. Our previous research has established that the, B2-subunit of the nicotinic acetylcholine receptor (nAChR) is involved in several behavioral and neurochemical actions of nicotine relevant to addiction, however, these nAChRs are widely expressed and the precise anatomical/cellular basis for these behaviors is not known. In this proposal, traditional pharmacological, biochemical and behavioral approaches will be combined with novel transgenic and knockout technologies in order to test the hypothesis that activation of high affinity nAChRs in discrete brain subregions is critical for mediating these distinct behavioral actions of nicotine. Newly generated lines of mice with conditional expression of the, B2-subunit in the mesostriatal system, the cortico-thalamic system or in neurons that bind alpha-conotoxin MII will be used, along with previously characterized knockout mice lacking the, B2-subunit, to identify brain regions that mediate the behavioral and pharmacological actions of nicotine associated with dependence and withdrawal. Aim 1 of this project will be to identify the brain areas where the highest affinity nAChRs must be expressed in order to restore nicotine-induced locomotor activation or nicotine withdrawal. Aim 2 will identify the anatomical sites where nAChRs can mediate increased extracellular dopamine release in the nucleus accumbens and/or prefrontal cortex. Aim 3 will use Western blotting to identify changes in levels of signaling proteins associated with the behavioral and neurochemical actions of nicotine identified in Aims 1 and 2. In addition, immediate early gene expression will be used as a marker of neuronal activation to identify brain regions activated during nicotine-induced hyperlocomotion or withdrawal. Knockout mice lacking the B2- subunit will serve as a control to show which pharmacological and biochemical actions of nicotine are mediated through the high affinity subclass of nAChR and the newly developed lines of transgenic mice conditionally expressing the, B2-subunit will then be used to identify brain regions critical for these effects. These experiments will determine whether the meso-cortico-limbic pathway is necessary for the development of nicotine dependence and withdrawal but should also identify other brain areas that contribute to nicotine actions. In addition, these experiments will help identify the molecular mechanisms and signaling pathways involved in the switch from nicotine use to dependence.